FGF having enhanced stability

ABSTRACT

Methods are provided that exploit thermostable FGF-1 proteins for support of human pluripotent stem cell cultures. Also provided are compositions containing thermostable FGF-1 for culturing of human pluripotent stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/717,055 filed Dec. 17, 2012 to issue as U.S. Pat. No. 9,023,644 onMay 5, 2015, which claims priority to U.S. Provisional PatentApplication No. 61/576,803 filed Dec. 16, 2011, each of which isincorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under ES017166 andGM081629 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The invention relates generally to methods and compositions forculturing human pluripotent stem cells, and, more particularly, tomethods and compositions having thermostable fibroblast growth factor(FGF) proteins for improved culture efficiency.

Human pluripotent cells, such as human embryonic stem (ES) cells andhuman induced pluripotent stem (iPS) cells have the potential toproliferate indefinitely and to differentiate into cells of all threegerm layers (Lowry et al., PNAS 105: 2883-2888, 2008; Park et al.,Nature 451:141-U141, 2008; Reubinoff et al., Nat. Biotechnol.18:399-404, 2000; Takahashi et al., Cell 131:861, 872, 2007; Thomson etal., Science 282:1145-1147, 1998; Yu et al., Science 318:1917-1920,2007). These properties make human pluripotent cells invaluable forstudying embryogenesis, for drug discovery, and for clinicalapplications.

Current in vitro culture methods for human ES and iPS cells require theaddition of exogenous growth factors (Amit et al., Nat. Rev. DrugDiscov. 8:235-253, 2004; Ludwig et al., Nat. Biotechnol. 24:185-187,2006; Sato et al., Nat. Med. 10:55-63, 2004; Vallier et al., J. CellSci. 118:4495-4509, 2005; Wang et al., Blood 110:4111-4119, 2007). It ispresently thought that three growth factors are sufficient to maintainpluripotency and self-renewal of human ES and iPS cells throughactivation of the FGF, TGF/Nodal, and Insulin/IGF pathways (Bendall etal., Nature 448:1015-1021 (2007); Eiselleova et al., Stem Cells27:1847-1857 (2009); Vallier et al., J. Cell Sci. 118:4495-4509 (2005)).

The FGF pathway has been implicated in many stages of human pluripotentcell regulation, cell survival, proliferation, pluripotency, and lineagedetermination during differentiation (Eiselleova et al., Stem Cells27:1847-1857, 2009; Lanner and Rossant, Development 137:3351-3360, 2010;Levenstein et al., Stem Cells 24:568-574, 2006; Vallier et al., J. CellSci. 118:4495-4509, 2005; Xu et al., Nat. Meth. 2:185-190, 2005). TheFGF pathway is activated through the binding of FGF proteins to FGFreceptors, which triggers MAP kinase cascades to regulate downstreamevents (Lanner and Rossant, 2010).

FGF-1-9 are 150-250 amino acid proteins with approximately 30-70%sequence homology in their 120-amino acid core region (Ornitz et al.,Genome Biol. 2:3005.1-3005.12 (2001); Itoh et al., Trends Genet.20:563-569 (2004)). Because of their substantial sequence homology, newmembers of the FGF family were identified in several species, fromCaenorhabditis elegans to Homo sapiens (Itoh et al.), usinghomology-based methods. Twenty-two FGF family members have beenidentified in humans and mice (Ornitz et al., 2001; Itoh et al., 2004).

While different FGF proteins are used for various applications in cellculture, qualitative differences in cell responses elicited by thevarious FGF proteins remain ill-defined and poorly understood. Thefunctional difference between FGF proteins that can and cannot supporthuman pluripotent stem cells might be attributable to (1) the differentaffinity of the various FGF proteins to each of the four FGF receptors(FGFR) that lead to the activation of specific pathways (Eswarakumar etal., Cytokine Growth Factor Rev. 16:139-149, 2005; Mohammadi et al.,Cytokine Growth Factor Rev. 16:107-137, 2005; Zhang et al., J. Biol.Chem. 281:15694-15700, 2006); and (2) the differential expression ofFGFs and FGFRs in specific tissues (Beenken and Mohammadi, Nat. Rev.Drug Discov. 8:235-253, 2009). However, these factors insufficientlyexplain the functional differences between FGF-2 and other FGF proteinsin human ES cell culture.

FGF-2 is routinely used for human ES and iPS cell culture (Levenstein etal., Stem Cells 24:568-574, 2006). Interestingly, FGF-1 does not supporthESC pluripotency or cell survival, even though FGF-1 targets the sameset of receptors as FGF-2 (Zhang et al., J. Biol. Chem. 281:15694-15700,2006).

While FGF-2 supports pluripotency in defined long-term human pluripotentcell cultures, high FGF-2 concentrations (e.g., 100 ng/ml) are required,which significantly increases culture cost. It has been suggested thathigh FGF-2 concentrations might be required to satisfy specificdose-dependent signaling thresholds, and to overcome obstacles such asprotein degradation (Levenstein et al., Stem Cells 24:568-574, 2006).Heparin and heparan sulfate can facilitate binding between FGF and FGFRto stimulate downstream activation (Levenstein et al., Stem Cells26:3099-3107, 2008; Mohammadi et al., Curr. Opin. Struct. Biol.15:506-516, 2005). Heparin and heparan sulfate promote pluripotency(Fume et al., PNAS 105:13409-13414, 2008; Levenstein et al., Stem Cells26:3099-3107, 2008), although it is unclear whether they do so via theFGF pathway. Heparin appears to increase the stability of FGF-1 andmight be important in the formation of FGF-1-FGFR complexes (Zakrzewskaet al., J. Biol. Chem. 284:25388-25403 (2009)). While FGF-2 fromzebrafish is capable of supporting self-renewal (Ludwig et al., Nat.Meth. 3:637-646, 2006), effective mammalian FGFs that can be used as analternative to mammalian wild type FGF-2 are not known. Thus, there is aneed in the art for more efficient growth factors that can support humanpluripotent stem cells in culture.

BRIEF SUMMARY

In a first aspect, the present invention is summarized as a method forculturing human pluripotent stem cells, comprising the step of culturinga human pluripotent stem cell in a medium comprising at least onethermostable fibroblast growth factor-one (FGF-1) that comprises aminoacid substitutions at positions corresponding to positions 40, 47, and93 of wildtype truncated FGF-1 (SEQ ID NO:2), wherein the mediumcomprising the thermostable FGF-1 is characterized by an enhancedability to support pluripotency relative to a medium comprisingtruncated wild type FGF 1 (SEQ ID NO:2).

In some embodiments of the first aspect, the amino acid sequence of thethermostable FGF-1 further comprises an amino acid substitution withinthe heparin binding domain. In some embodiments, the amino acidsubstitution within the heparin binding domain is at position 112 of SEQID NO:2.

In some embodiments of the first aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:3. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:3.

In further embodiments of the first aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:4. In some embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:4.

In some embodiments of the first aspect, the human pluripotent stemcells to be cultured are human embryonic stem cells or human inducedpluripotent stem cells.

In some embodiments of the first aspect, the medium to be used furthercomprises heparin.

In some embodiments of the first aspect, the concentration of thethermostable FGF-1 in the medium is less than 40 ng/ml (e.g., 10 ng/mlor less).

In a second aspect, the present invention is summarized as afully-defined medium suitable for culturing human pluripotent cells inan undifferentiated state, the defined medium comprising a thermostablefibroblast growth factor-one (FGF-1) that comprises amino acidsubstitutions at positions corresponding to positions 40, 47, and 93 ofwildtype truncated FGF-1 (SEQ ID NO:2).

In some embodiments of the second aspect, the thermostable FGF-1,present in the fully defined medium, further comprises an amino acidsubstitution corresponding to position 112 of the wild type FGF-1 (SEQID NO:2).

In some embodiments of the second aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:3. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:3.

In some embodiments of the second aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:4. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:4.

In some embodiments of the second aspect, the thermostable FGF-1 isprovided in the fully defined medium at a concentration of less than 40ng/ml (e.g., 10 ng/ml or less).

In a third aspect, the present invention is summarized as a compositioncomprising: a human pluripotent stem cell and a culture mediumcomprising a thermostable fibroblast growth factor-one (FGF-1) thatcomprises amino acid substitutions at positions corresponding topositions 40, 47, and 93 of wildtype truncated FGF-1 (SEQ ID NO:2), andwherein the culture medium comprising the thermostable FGF-2 is suitablefor culturing the human pluripotent cell in an undifferentiated state,and is characterized by an enhanced ability to support pluripotencyrelative to a medium comprising a truncated wild type FGF-1 (SEQ IDNO:2).

In some embodiments of the third aspect, the thermostable FGF-1 in thecomposition further comprises an amino acid substitution within theheparin binding domain (e.g., at a position corresponding to position112 of a truncated wild type FGF-1 (SEQ ID NO:2).

In some embodiments of the third aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:3. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:3.

In some embodiments of the third aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:4. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:4.

In a fourth aspect, the present invention is summarized as a method fordifferentiating a human pluripotent stem cell into a mesoderm lineagecell, where the method comprises the step of culturing the humanpluripotent stem cell in a medium until the cell expresses mesodermlineage markers, wherein the medium comprises BMP4 and a thermostablefibroblast growth factor-one (FGF-1) that comprises amino acidsubstitutions at positions corresponding to positions 40, 47, and 93 ofwildtype truncated FGF-1 (SEQ ID NO:2), and wherein the mediumcomprising the BMP 4 and the thermostable FGF 1 is characterized by anenhanced ability to support differentiation of a human pluripotent stemcell into a mesoderm lineage cell relative to a medium comprising BMP4and a truncated wild type FGF-1 (SEQ ID NO:2).

In some embodiments of the fourth aspect, the thermostable FGF-1 furthercomprises an amino acid substitution at a position corresponding toposition 112 of the wild type truncated FGF-1 (SEQ ID NO:2).

In some embodiments of the fourth aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:3. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:3.

In some embodiments of the fourth aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:4. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:4.

In a fifth aspect, the present invention is summarized as a method forreprogramming a human somatic cell into a human pluripotent cell, wherethe method comprises culturing the somatic cell in a medium until thecell expresses markers indicative of a human pluripotent cell, whereinthe medium comprises water, salts, amino acids, vitamins, a carbonsource, insulin, selenium and at least one thermostable fibroblastgrowth factor-one (FGF-1) that comprises amino acid substitutions atpositions corresponding to positions 40, 47, and 93 of wildtypetruncated FGF-1 (SEQ ID NO:2), and wherein the medium comprising thethermostable FGF-1 is characterized by an enhanced ability to supportpluripotency relative to a medium comprising a truncated wild type FGF-1(SEQ ID NO:2).

In some embodiments of the fifth aspect, the thermostable FGF-1 furthercomprises an amino acid substitution at a position corresponding toposition 112 of the wild type FGF-1 (SEQ ID NO:2).

In some embodiments of the fifth aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:3. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:3.

In some embodiments of the fifth aspect, the thermostable FGF-1comprises the amino acid sequence of SEQ ID NO:4. In other embodiments,the amino acid sequence of the thermostable FGF-1 consists of the aminoacid sequence of SEQ ID NO:4.

In further embodiments of the fifth aspect, the medium is serum-free. Inother embodiments of the fifth aspect, the medium further comprisesheparin.

The methods and compositions described herein are useful in a variety ofapplications, such as maintaining and passaging a viable population ofhuman pluripotent stem cells, reprogramming human somatic cells intopluripotent stem cells, or differentiating human pluripotent stem cellsalong certain lineages, e.g., the mesodermal lineage, for which FGF isan important morphogen.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to preclude theinvention from covering modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 A-F illustrates that thermostability of FGF affects its abilityto stimulate ERK phosphorylation. FIG. 1A shows that ERK phosphorylationcorrelates with the activation of FGF receptors in human ES cells. H1cells were incubated in E₈ media (Chen et al., Nat. Meth. 8:424-429,2011) (100 ng/ml FGF-2 and 2 ng/ml Tgfβ for 30 minutes with drugtreatments (10 μM SU5402-FGFR inhibitor or 10 μM SB43542-TGFβ inhibitor,or both). Proteins were harvested to analyze ERK1/2 phosphorylation(pERK1/2) by western blot. FIG. 1B shows that inhibition of ERKphosphorylation suppresses NANOG expression; NANOG expression wasmeasured after three days of incubation. FIG. 1C shows screening forFGFs supporting sustained ERK phosphorylation. H1 ES cells were platedinto basic media (E₈ media without TGFβ) with different FGFs (100 ng/ml)for 24 hours, and proteins were then collected to detect ERKphosphorylation. FIG. 1D shows screening for FGFs supportingpluripotency. H1 cells were maintained in the same media as (C) forthree days, cells were harvested to measure the expression of NANOG byRT-qPCR. GAPHD was used as control. FIG. 1E shows screening for FGFsthat stimulate ERK phosphorylation in short exposure. Media used in (C)were applied for 15 minutes on FGF-starved ES cells before proteins werecollected to analyze for ERK phosphorylation. FIG. 1F shows howthermostability of FGF affects activation of ERK phosphorylation. Mediaused in (C) were incubated at 37° C. for 6 hours, and then applied for15 minutes on FGF-starved ES cells, before proteins were collected forERK phosphorylation analysis.

FIG. 2A-C illustrates dynamic regulation to maintain FGF pathwayactivation at relatively low level. FIG. 2A shows that ERKphosphorylation decreases after initial activation. FGF-2 (100 ng/ml)was added to FGF-starved ES cells, and proteins were collected atspecific time points for western blot. ERK phosphorylation wassignificantly lower than previous time points. FIG. 2B shows that therewas no significant loss of FGF-2 activity in media at 12 hours. Growthmedia was collected from the cell culture and was applied to FGF-starvedcells for 15 minutes and proteins were collected for western blot. FIG.2C shows that ERK phosphorylation is controlled at a consistentlymoderate level in continuous FGF culture. FGF-2 was applied ontoFGF-starved and FGF-primed cells, and cells were harvested at specifictime points.

FIG. 3A-F Illustrates a thermostable FGF-1 mutant and its ability tosupport ERK phosphorylation and pluripotency. FIG. 3A shows that FGF-1is unstable at 37° C. Media with FGF-1 were incubated with heparin at37° C., and then applied for 15 minutes on FGF-starved ES cells atspecific time points, before proteins were collected to analyze for ERKphosphorylation. FIG. 3B shows FGF-1 protein mutated to increasethermostability or heparin affinity. The mutated amino acid isunderlined (Zakrzewska et al., J. Mol. Biol. 352:860-875, 2005;Zakrzewska et al., J. Biol. Chem. 284:25388-25403, 2009). FIG. 3C showsthat mutations to FGF-1 improved maintenance of ERK phosphorylation for24 hours. H1 ES cells were plated into basic media (E₈ media withoutTGFβ) with different FGFs (100 ng/ml) for 24 hours, and proteins werethen collected to detect ERK phosphorylation. FIG. 3D shows an FGF-1mutant FGF-1 4X (Q40P, S47I, H93G, with K112N) that is thermostable,while FGF-1 3X (Q40P, S47I, H93G) and FGF-2 were stabilized by heparin.Media with different FGFs were pre-incubated at 37° C. for 24 hours, andthen applied for 15 minutes on FGF-starved ES cells, before proteinswere collected to analyze for ERK phosphorylation. FIG. 3E shows thatmutated FGF-1 supports ES cell growth. H1 cells were maintained in E₈media with different FGFs, and cells were counted after 96 hours. FIG.3F shows that mutated FGF-1 supports pluripotency of human ES cells. H1cells were maintained in E8 media without TGFβ and different FGFs, andOCT4 expression was analyzed after 2 passages.

FIG. 4A-E illustrates stabilized FGF proteins. FIG. 4A shows that FGF-1and heparin help maintain ES cell morphology. H1 cells were cultured inspecific media for 5 days. FIG. 4B shows FGF-2 and FGF-1 derivativespurified from E. coli stained with Coomassie Blue following PAGEelectrophoresis. FIG. 4C shows that mutations did not affect FGFactivity significantly. Same amount of FGFs (10 ng/ml) was applied ontoFGF-starved cells for 15 minutes and protein was harvested to measureERK phosphorylation. FIG. 4D shows that FGF-1 3X cultured in E₈ (TGFβ)media sustained long-term ES cell culture. OCT4 staining was performedafter 3 passages. ES cells were maintained in FGF-1 3X media for 10passages, and cells maintained normal karyotype. FIG. 4E shows thatfull-length and truncated FGF-1s lost their activity after 6-hourincubation at 37° C., and heparin failed to preserve the activity. Mediawith full-length FGF-1 or truncated FGF-1 were treated with or withoutheparin at 37° C. for 6 hours and were then applied to FGF-starved EScells for 15 minutes. Protein was harvested to measure ERKphosphorylation.

FIG. 5 A-D illustrates that FGF stability affects differentiation andreprogramming. FIGS. 5A and 5B show that human ES cells differentiate inthe absence of TGFβ. H1 cells were maintained in E8 media with differentFGFs, and GATA2 and HAND1 were detected by RT-qPCR. GAPDH was used ascontrol, and expression level was normalized with the expression inFGF-1-WT. In FIGS. 5A and 5B, the left-hand column in each pairrepresents expression without heparin, and (the right-hand column ineach pair) represents expression with heparin. FIGS. 5C and 5D show thatFGF and BMP4 induce mesodermal specific differentiation in human EScells. Cells were incubated with BMP4 and different FGFs for 48 hours,and the expression of NANOG and T were detected by RT-qPCR. FIG. 5Edepicts human fibroblast cells maintained in defined fibroblast mediawith different FGFs; cells were counted after 120 hours. FIG. 5F depictshuman fibroblast cells reprogrammed in defined conditions with episomalvectors with different FGFs. iPS cells were scored after 25 days.

FIG. 6 illustrates that specific FGFs affect reprogramming efficiency infibroblasts. Foreskin fibroblasts were reprogrammed with viral-freeapproach (e.g., U.S. Patent Application Publication No. 2010/0184227).Different FGFs (100 ng/ml) were used to replace FGF-2 in culture mediaat each reprogramming stage. iPS colonies were scored 30 days after thetransfection of reprogramming factors.

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DETAILED DESCRIPTION

The invention relates to the use of variants of FGF-1 having enhancedthermostability to improve maintenance of pluripotency of cultured humanpluripotent stem cells, or to enhance their differentiation into certainlineages (e.g., mesoderm) when FGF is one of the morphogens used in thedifferentiation.

The present invention relates to the inventors' observation that proteinstability of FGF-family proteins plays an essential role in themolecule's ability to support human pluripotent cell cultures. Theinvention provides thermostable FGF-1 compositions, and methods tosupport human pluripotent stem cells in an undifferentiated state. Insome instances, the stability and effectiveness in supportingpluripotency of the thermostable FGF-1 can be further enhanced byheparin binding to the thermostable FGF-1.

Many FGF-family members, such as FGF-1, have repeatedly been shown tofail to maintain human pluripotent stem cells in culture, for unknownreasons. The invention presented herein demonstrates, for the firsttime, that thermostability of FGF-1 can be a determining aspect ofgrowth factor regulation in stem cell biology. The disclosed inventionalso demonstrates, for the first time, that amino acid substitutions inwild type FGF-1 that result in thermostable FGF-1 sequence variants havesuperior abilities to maintain pluripotency in long-term and underfeeder independent conditions. Unexpectedly, these FGF-1 sequencevariants can also act as effective morphogens to induce certainlineage-specific differentiation, and to enhance somatic cellreprogramming.

Thermoinstability of FGF-1 was not an obvious cause of wildtype FGF-1'sfailure to support human pluripotent stem cells in culture because therole of numerous characteristics of FGF-1 biology in maintainingpluripotency were unknown. FGF receptors in different cell types respondto FGF ligands differently. Without directly testing the influence ofindividual FGF variants on human ES cell growth and maintenance, theskilled artisan could not have predicted which FGF variants couldsupport pluripotency. Indeed, in view of the studies carried out onother cell types, and the fact that all FGF receptors are expressed inhES cells, the skilled artisan would have predicted that every FGFshould have the capacity to support pluripotency of hES or hiPS cells,which is not, in fact, the case.

I. Definitions

As used herein, “defined culture medium,” “defined medium,” or “fullydefined medium” refers to an essentially serum-free medium that hasknown quantities of all ingredients.

As used herein, “enhanced ability to support pluripotency” means that alower concentration of the thermostable FGF-1 can support maintenance ofpluripotency in vitro, compared to the concentration of other FGFs knownto support pluripotency, and compared to FGFs, such as wild type FGF-1,that do not support pluripotency. To confirm whether a thermostable FGFsupports pluripotency, 5 passages (˜20-30 days) in culture mediacontaining the thermostable FGF is usually required, wherein cellsmaintain morphology and genetic expression characteristic of humanpluripotent cells. For example, human pluripotent cells typicallyexhibit a round shape, large nucleoli and scant cytoplasm and expressOCT4. Thermostable FGF-1 can also support pluripotency of humanpluripotent cells in vitro for a longer period of time (e.g., greaterthan 48 hours) compared to wildtype FGF-2, which is effective for lessthan 24 hours.

As used herein, “iPS cells” refer to cells that are substantiallygenetically identical to their respective differentiated somatic cell oforigin and display characteristics similar to human ES cells, asdescribed herein. The cells can be obtained from various differentiatedsomatic cells, e.g., mononuclear blood cells, skin fibroblasts,keratinocytes, etc.

As used herein, “serum-free” means that neither the culture nor theculture medium contains serum or plasma, although purified or syntheticserum or plasma components (e.g., FGFs) can be provided in the culturein reproducible amounts as described below. For example, an essentiallyserum-free medium can contain less than about 1% serum or serumreplacement.

As used herein, thermostable “FGF-1,” refers to an FGF-1 having an aminoacid sequence that includes the amino acid sequence of SEQ ID NO:2 (atruncated wildtype FGF-1), but having amino acid substitutions at atleast three positions: 40, 47, and 93 relative to SEQ ID NO:2, andretaining the ability to stimulate ERK phosphorylation in humanpluripotent stem cells after a 24 hour incubation period with a givenamount of the thermostable FGF-1. In some cases, the thermostable FGF-1also includes a fourth amino acid substitution, which falls in theheparin binding domain of truncated FGF-1, e.g., position 112.

As used herein, “wild type amino acid sequence” refers to the mostcommon amino acid sequence among members of a species.

As used herein, “thermostable FGF-1” refers to an FGF-1 protein havingan altered amino acid sequence relative to the wild type FGF-1 sequencethat is also more stable than wild type FGF-1 under human pluripotentstem cell culture conditions. As disclosed herein, binding of wild typeFGF-1 to heparin or FGF-binding protein (FGFP) enhances itsthermostability but fails to render FGF-1 capable of long-termundifferentiated human pluripotent cell culture. In the context of thisapplication, a wild type FGF-1 protein bound to heparin, even if morestable to heat than unbound FGF-1, is not encompassed by the term“thermostable FGF.”

II. Methods

The invention provides a method for culturing human pluripotent stemcells in culture. Human pluripotent stem cells, such as human embryonicstem cells or induced pluripotent stem cells, are cultured in a mediumcontaining a thermostable FGF-1.

In some embodiments, human pluripotent stem cells, such as humanembryonic stem cells or human induced pluripotent cells, are cultured ina medium containing at least one thermostable FGF-1. The thermostableFGF-1 sequence variants described herein are characterized by anenhanced ability, compared to the respective wild type FGF-1 protein, tosupport pluripotency of cultured human pluripotent stem cells over time,such as several weeks and passages, in culture. Methods for introducingsingle or multiple changes into the amino acid sequence of an FGFprotein are well known in the art (e.g., Kim et al. J. Mol. Biol.328(4): 951-961 (2003); Brych et al. J. Mol. Biol. 344(3): 769-780(2004); Lee et al., J. Mol. Biol. 393(1): 113-127 (2009); Zakrzewska etal., J. Mol. Biol. 352(4): 860-875 (2005); Zakrzewska et al., ProteinEng. Des. SeI17(8): 603-611 (2004); Zakrzewska et al., J. Biol. Chem.284(37): 25388-25403 (2009); U.S. Pat. No. 7,659,379, each of which isincorporated herein by reference as if set forth in its entirety).

In some embodiments, the thermostable FGF-1 used in the method comprisesthe amino acid sequence of a thermostable fibroblast growth factor-one(FGF-1) that comprises amino acid substitutions at positionscorresponding to positions 40, 47, and 93 of wildtype truncated FGF-1(SEQ ID NO:2), wherein the medium comprising the thermostable FGF-1 ischaracterized by an enhanced ability to support pluripotency relative toa medium comprising truncated wild type FGF-1 (SEQ ID NO:2). Thenomenclature of the FGF-1 sequence variants disclosed herein follows thetruncated FGF-1 naming convention, following the numbering conventionestablished by Gimenez-Gallego et al., Biochem. Biophys. Res. Comm. 128611-617 (1986).

In some embodiments, the thermostable FGF-1 to be used includes theamino acid sequence shown in SEQ ID NO:3 (“FGF1-3X”), which containsQ40P, S47, and H93 mutations. Optionally, the thermostable FGF-1 to beused in the method can further include a single amino acid substitutionwithin the heparin binding domain of the thermostable FGF-1. The skilledartisan would have found it counterintuitive to introduce a mutation inthe heparin binding domain because binding to heparin enhances stabilityof FGF-1 and -2 at 37° C. In some embodiments, the amino acidsubstitution in the heparin binding domain is at position 112 relativethe amino acid sequence of truncated wild type FGF-1 (position 127 infull length, wildtype FGF-1), as shown in SEQ ID NO:2. K112 (K127 infull length FGF-1) is the amino acid that significantly contributes toheparin binding (Zakrzewska et al., J. Biol. Chem. 284:25388-25403,2009). In one embodiment, the amino acid substitution at position 112 isa K112N substitution.

In other embodiments, the thermostable FGF-1 to be used in the methodincludes the amino acid sequence shown in SEQ ID NO:4 (“FGF1-4X”), whichcontains four amino acid substitutions: Q40P, S47, H93, and K112N. Insome embodiments, the thermostable FGF-1 to be used includes the aminoacid sequence of SEQ ID NO:3 or SEQ ID NO:4, but is greater in lengththan either of these amino acid sequences (e.g., an N-terminal orC-terminal fusion polypeptide). For example, in some cases, thethermostable FGF-1 to be used, in addition to the amino acid sequence ofSEQ ID NO:3 or SEQ ID NO:4 also includes the full length amino terminalsequence of human wildtype FGF-1. polypeptide thermostable FGF-1 to beused is a full length human FGF-1 comprising the amino acid sequence ofSEQ ID NO:3. include a mutation In one embodiment, the thermostableFGF-1. In other embodiments, the amino acid sequence of the thermostableFGF-1 consists only of the amino acid sequence of the truncated humanFGF-1 sequence variant of SEQ ID NO:3 or SEQ ID NO:4.

In some embodiments, in addition to a thermostable FGF-1, heparin isalso included in the medium used in the culture method. A suitableconcentration of heparin ranges from about 50 ng/ml to about 200 ng/ml,e.g., about 60 ng/ml, 75 ng/ml, 90 ng/ml, 100 ng/ml, 125 ng/ml, 150ng/ml, 175 ng/ml, or another concentration of heparin from about 50ng/ml to about 200 ng/ml.

A medium used in the methods to culture and maintain human pluripotentstem cells, as described herein, can be any medium that supports humanpluripotent cells in culture (e.g., Chen et al., Nat. Meth. 8:424-429,2011 or any of the commercial media mentioned herein), but in which athermostable FGF-1 is substituted, at least in part, for an FGF (e.g.,FGF-2) normally used in the completed culture medium (e.g., mTeSR®). Insome embodiments, thermostable FGF-1 is the only FGF-1 used in themedium used in the culturing method, i.e., it is substituted incommercial media that are typically pre-formulated with wild type FGF-2.In other embodiments, the medium to be used might may contain 20%, 40%,60%, 80% or 99% thermostable FGF-1 (e.g., thermostable FGF1-4X) and thebalance being wild type FGF-2. Preferably, the medium is fully defined.

Preferably, the thermostable FGF-1 concentrations used to supportpluripotency are lower than for wild type FGF-1 proteins. For example, athermostable FGF-1 supports pluripotency at concentrations lower thanthose required for FGF-1 bound to heparin or for FGF-2. Specifically, anexemplified thermostable FGF-1 sequence variant, FGF1-4X (SEQ ID NO:4),with 4 amino acid mutations, Q40P, S47I, H93G, and K112N relative to SEQID NO: 2, as exemplified herein, can support self-renewal of humanpluripotent ES and iPS cells at concentrations 4 to 10-fold lower thanthose ordinarily used for FGF-2 (about 40-100 ng/ml).

In some embodiments of the invention, human pluripotent cells arecultured with a thermostable FGF-1 at a concentration of 40 ng/ml orless, preferably 10 ng/ml or less, 3 ng/ml or less, or 1 ng/ml or less.It is contemplated that culture conditions including a thermostableFGF-1 (e.g., FGF1-3X or FGF1-4X) at a concentration of 40 ng/ml or less,preferably 10 ng/ml, 3 ng/ml, or 1 ng/ml or less, are sufficient formaintaining human pluripotent cells and for reprogramming somatic cellsinto induced human pluripotent cells. In some embodiments, FGF1-3X orFGF1-4X are included at a concentration of about 10 ng/ml to about 50ng/ml, 10 ng to about 30 ng/ml, 3 ng/ml to about 10 ng/ml, 1 ng/ml toabout 3 ng/ml, or about 0.2 ng/ml to about 1 ng/ml.

To confirm that a particular thermostable FGF-1 sequence variant cansupport pluripotency, human pluripotent cells are cultured in a mediumcontaining the thermostable FGF-1 for at least 5 passages (˜20-30 days)and then evaluated for pluripotency. Criteria for evaluatingpluripotency of human pluripotent stem cells are known in the art, andinclude, but are not limited to, expression of Oct4 and Nanog mRNA andprotein, and suppression of differentiation markers (e.g., Hand1 andGata). The function of FGF-2 in human pluripotent stem cells can beconveniently assessed using a biochemical endpoint such as ERKphosphorylation. Measuring increased ERK phosphorylation in response toFGF stimulation, as described herein, provides a rapid measure of theability of a thermostable FGF-2 to support in vitro pluripotency. Othermethods of assessing pluripotency are also suitable.

The invention is also directed at a method for reprogramming humansomatic cells into human induced pluripotent (iPS) cells in culture.Somatic cells, such as foreskin fibroblast cells, are cultured in amedium containing a thermostable FGF-1, as described herein, e.g., inthe presence of FGF1-4X, FGF1-3X, or a combination thereof. Suitableculture media (buffered to a pH of about 7.4) for reprogramming humansomatic cells, in addition to a thermostable FGF-1, as described herein,include water, salts, amino acids, vitamins, a carbon source, insulin,and selenium. The medium can be any medium that supports humanpluripotent cells in culture (e.g., Chen et al., Nat. Meth. 8:424-429,2011). Preferably, the medium is fully defined. The medium can be any ofthe above-mentioned media that support pluripotency of human pluripotentstem cells. Preferably, the medium to be used is fully defined, e.g., E8medium. Human somatic cells can be reprogrammed using methods known inthe art (e.g., Patent Application Publication Nos. 2008/0233610 and2010/0184227, each incorporated herein by reference in its entirety asif set forth herein. A medium containing a thermostable FGF-1 is suitedfor use in other reprogramming methods, such as those mentioned below(and, likewise, each incorporated by reference herein in its entirety):Adenoviral vector reprogramming (Zhou and Freed, Stem Cells 27:2667-2674, 2009); Sendai virus reprogramming (Fusaki et al., Proc JpnAcad 85: 348-362, 2009); polycistronic minicircle vector reprogramming(Jia et al., Nat Methods 7: 197-199, 2010); piggyBac transposonreprogramming (Woltjen et al., Nature 458: 766-770, 2009; Yusa et al.,Nat Methods 6: 363-369, 2009); recombinant proteins for reprogramming(Zhou et al., Cell Stem Cell 4: 381-384, 2009); whole cell extractsisolated from human ES cells (Cho et al., Blood 116: 386-395, 2010) orgenetically engineered HEK293 cells (Kim et al., Cell Stem Cell 4:472-476, 2009); small molecules to replace individual reprogrammingfactors (Desponts and Ding, Methods Mol Biol 636: 207-218, 2010; Li andDing, Trends Pharmacol Sci 31: 36-45, 2010). Suitable human somaticcells for reprogramming include, but are not limited to, bloodmononuclear cells, skin-derived fibroblasts, and keratinocytes.

The invention is also directed to a method for differentiating a humanpluripotent stem cell into a mesoderm lineage cell, where the methodincludes culturing human pluripotent stem cells in a medium until thecultured cells express mesoderm lineage markers, wherein the mediumcomprises BMP4 and a thermostable FGF-1 comprising the amino acidsequence of a thermostable fibroblast growth factor-one (FGF-1) thatcomprises amino acid substitutions at positions corresponding topositions 40, 47, and 93 of wildtype truncated FGF-1 (SEQ ID NO:2), andwherein the medium comprising the BMP4 and the thermostable FGF-1 ischaracterized by an enhanced ability to support differentiation of ahuman pluripotent stem cell into a mesoderm lineage cell relative to amedium comprising BMP4 and a truncated wild type FGF-1 (SEQ ID NO:2).

Suitable media for mesodermal differentiation, include any of thethermostable FGF-1-containing media described herein (e.g., E8 mediumplus FGF1-4X) plus an appropriate concentration of BMP4, which may rangefrom about 2 ng/ml to about 10 ng/ml, e.g., about 3 ng/ml, 5 ng/ml, 7ng/ml, 8 ng/ml or another concentration of BM4 from about 2 ng/ml toabout 10 ng/ml. Markers for assessing mesoderm lineage differentiationinclude, but are not limited to, Brachyury (T), WNT3, and MIXL1.

III. Compositions

The invention is also directed to a fully-defined medium suitable forculturing human pluripotent stem cells in an undifferentiated statethroughout several passages, the medium containing at least onethermostable FGF-1 having enhanced ability to support pluripotency, asdescribed herein. Culture conditions that permit the long-term cultureof undifferentiated human pluripotent cells in a defined mediumsupplemented with high concentrations of FGF-2, e.g., 100 ng/ml, areknown in the art (e.g., Ludwig et al., Nat. Methods 3:637-646 (2006),incorporated herein by reference as if set forth in its entirety). It isspecifically contemplated that the fully-defined medium described hereincontains at least one thermostable FGF-1 at a concentration lower thanthat required of wild type FGF-2 protein, wherein the lowerconcentration is at least 5% lower than that required of wild type FGF-2protein, preferably at least 10% lower than that required of wild typeFGF-2 protein, more preferably at least 15%, 20%, 30%, 40%, 50%, or atleast 60% lower than that required of wild type FGF-2 protein.

In some embodiments, the medium contains a thermostable FGF-1 insteadof, or in addition to, wild type FGF-2. In a preferred embodiment, thismedium is suitable for the derivation of human ES cell lines andreprogramming of somatic cells. Use of this medium in methods fordifferentiating human pluripotent stem cells, such as into cells of themesoderm lineage, and in methods of reprogramming somatic cells into iPScells, is specifically contemplated, as described herein.

In some embodiments, the fully defined medium includes a thermostablefibroblast growth factor-one (FGF-1) that comprises amino acidsubstitutions at positions corresponding to positions 40, 47, and 93 ofwildtype truncated FGF-1 (SEQ ID NO:2), wherein the medium comprisingthe thermostable FGF-1 is characterized by an enhanced ability tosupport pluripotency relative to a medium comprising truncated wild typeFGF-1 (SEQ ID NO:2).

In some embodiments, the fully defined medium includes a thermostableFGF-1 comprising the amino acid sequence shown in SEQ ID NO:3(“FGF1-3X”), which contains Q40P, S47, and H93 mutations. Optionally,the medium can include a thermostable FGF-1 that has a single amino acidsubstitution within the heparin binding domain of the thermostableFGF-1. In some embodiments, the amino acid substitution in the heparinbinding domain is at position 112 relative the amino acid sequence oftruncated wild type FGF-1 (position 127 in full length, wildtype FGF-1),as shown in SEQ ID NO:2. In one embodiment, the amino acid substitutionat position 112 is a K112N substitution.

In other embodiments, the thermostable FGF-1 to be used in the methodincludes the amino acid sequence shown in SEQ ID NO:4 (“FGF1-4X”), whichcontains four amino acid substitutions: Q40P, S47, H93, and K112N. Insome embodiments, the thermostable FGF-1 in the fully defined mediumincludes the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, but isgreater in length than either of these amino acid sequences (e.g., anN-terminal or C-terminal fusion polypeptide). For example, in somecases, the thermostable FGF-1 to be used, in addition to the amino acidsequence of SEQ ID NO:3 or SEQ ID NO:4 also includes the full lengthamino terminal sequence of human wildtype FGF-1 (i.e., amino acids 3-21are retained). polypeptide thermostable FGF-1 to be used is a fulllength human FGF-1 comprising the amino acid sequence of SEQ ID NO:3.include a mutation In one embodiment, the thermostable FGF-1. In otherembodiments, the amino acid sequence of the thermostable FGF-1 consistsonly of the amino acid sequence of the truncated human FGF-1 sequencevariant of SEQ ID NO:3 or SEQ ID NO:4.

In some embodiments, where the medium contains a thermostable FGF1-3X(SEQ ID NO:3), heparin is also included in the medium. A suitableconcentration of heparin ranges from about 50 ng/ml to about 200 ng/ml,e.g., about 60 ng/ml, 75 ng/ml, 90 ng/ml, 100 ng/ml, 125 ng/ml, 150ng/ml, 175 ng/ml, or another concentration of heparin from about 50ng/ml to about 200 ng/ml.

The invention is also directed at a composition that contains humanpluripotent stem cells (e.g., human embryonic stem cells or humaninduced pluripotent stem cells), and any of the thermostableFGF-1-containing media described herein that is suitable for maintainingpluripotency of human pluripotent stem cells.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples. All papers and patents disclosed hereinare hereby incorporated by reference as if set forth in their entirety.

EXAMPLES Example 1 Variable FGF Pathway Activation in Human PluripotentStem Cells by FGF Family Members

Cell Culture

Human ES cells were maintained in specific media on matrigel-coatedtissue culture plates essentially as described previously (Ludwig etal., Nat. Meth. 3:637-646, 2006). Cells were passaged with EDTAessentially as described previously (Chen et al., Cell Stem Cell7:240-248, 2010). Briefly, cells were washed twice with PBS/EDTA medium(0.5 mM EDTA in PBS, osmolarity 340 mOsm), then incubated with PBS/EDTAfor 5 minutes at 37° C. The PBS/EDTA was removed, and the cells werewashed swiftly with a small volume of medium.

Cell Growth Measurement

Cell growth was analyzed essentially as described previously (Chen etal., Cell Stem Cell 7:240-248, 2010). E8 cell culture medium was usedfor cell growth experiments (Chen et al., Nat. Meth. 8:424-429, 2011).All experiments were performed in triplicate using 12-well plates. Priorto the addition of cells, 500 μl medium was loaded into each well. Cellswere dissociated for 5 minutes or until fully detached from the platewith TrypLE (LIFE TECHNOLOGIES), which was subsequently neutralized withequal volumes of media. The cells were counted, washed, and diluted toconcentrations of 100,000 to 300,000 cells/ml and 100 μl of the cellsolution was added into each well. At various time points, cells wereagain dissociated with 0.4 ml TrypLE, neutralized with equal volumes of10% FBS in DMEM, and counted using flow cytometry. Approximately 5000count-bright beads (LIFE TECHNOLOGIES) were added to each sample as aninternal control and 200 beads were counted for each sample. Forproliferation experiments, media were changed daily up to the day ofanalysis, and cells were counted as described above.

FGF Expression and Purification

FGF proteins were expressed in ROSETTA™ 2 (DE3) pLysS cells (NOVAGEN®)using MAGICMEDIA® (LIFE TECHNOLOGIES) at 37° C. for 24 hours. FGFproteins were purified essentially as described by Wiedlocha et al.,Mol. Cell. Biol. 16(1): 270-280 (1996), incorporated herein by referenceas if set forth in its entirety. Briefly, bacterial pellets weresonicated and centrifuged. The clear supernatant was applied to aheparin cartridge (BIO-RAD) equilibrated with 0.5 M NaCl in 20 mM sodiumphosphate (pH 7.5)-1 mM EDTA-1 mM dithiothreitol. Fusion proteins wereeluted with 1 M NaCl in the same buffer and dialyzed against 20 mMsodium phosphate (pH 8.0)-1 mM EDTA-1 mM dithiothreitol. Subsequently,the fusion proteins were applied to a Q cartridge (BIO-RAD) and elutedwith a linear NaCl gradient in the same buffer.

FGF-1 derivatives were generated in the form of truncated FGF-1, whereinthe first 19 of 21 (positions 3-21) amino acid residues were deleted.

Pluripotency of human ES and iPS cells is supported by FGF andTGFβ/NODAL pathways (Vallier et al., J. Cell Sci. 118:4495-4509, 2005).FGF-2, not TGFβ, stimulates MAP kinase ERK1/2 phosphorylation aftershort-term incubation (15 minutes) (FIG. 1A). At the same time, ERKinhibition suppresses expression of pluripotency marker genes, such asNANOG (FIG. 1B). Cell culture experiments showed that an increase inextracellular signal-regulated kinase (ERK) phosphorylation in ES cellscan be used as reliable indicator of FGF pathway activation. Using ERKphosphorylation and NANOG expression to determine the function of thevarious FGF family members on human pluripotent stem cells revealed thatonly FGF-2, FGF-4, FGF-6, and FGF-9 were able to sustain strong ERKphosphorylation after 24 hours in cell culture (FIG. 1C). These resultswere consistent with NANOG expression in response to the various FGFs inES cells (FIG. 1D). However, 15 minute incubations with the various FGFfamily members led to different ERK phosphorylation patterns (FIG. 1E).Strong ERK phosphorylation induced by FGF-1, FGF-2, FGF-4, FGF-6, andFGF-9 correlated with the respective FGF's ability to bind to FGF-1R.FGF-1 was able to induce ERK phosphorylation but this activity was lostwithin about six hours of incubation at 37° C. (FIG. 1F). These resultsindicate that FGF-1 is not as stable at 37° C. relative to other FGFproteins, such as FGF-2 and FGF4, which might contribute to itsinability to maintain ERK phosphorylation and pluripotency in human EScells.

Example 2 Thermostable FGF-1 Supports Pluripotency of Human ES Cells inVitro

To determine how the function of FGF proteins can be affected bythermostability, above and beyond ligand-specificity, FGF-1 was used asa model protein. FGF-1 is not stable at 37° C. and does not supportself-renewal of human ES cells (FIG. 1). Frequent addition of mediumcontaining FGF-1 and addition of heparin significantly improvedshort-term cell culture (FIG. 2A). Nevertheless, FGF-1 activity waslargely lost after 6 hours at 37° C., even in the presence of heparin(FIG. 3A). Truncated FGF-1 protein, which is widely available, was alsounstable at 37° C. and exhibited thermostability dynamics similar tofull length FGF1 (FIG. 4E), suggesting that the N-terminal sequence ofFGF-1 does not play an important role in thermostability.

To determine if FGF-1 stability contributes, at least in part, to therapid loss of FGF-1 activity, several FGF-1 mutants were analyzed fortheir ability to support ES cells. The amino acid sequence of thesemutant FGF-1 proteins affects their stability or their ability to bindheparin but not their ability to bind FGF receptors (Zakrzewska et al.,J. Mol. Biol. 352:860-875, 2005; Zakrzewska et al., J. Biol. Chem.284:25388-25403, 2009) (FIG. 3B and FIG. 4B). The Lysine residue atposition 112 (K112) of FGF-1 is important for heparin binding. An aminoacid other than lysine at position 112, e.g., asparagine (K112N),results in weak heparin affinity. Glutamine at position 40 (Q40), serineat position 47 (S47), and histidine at position 93 (H93) of FGF-1 arenot exposed to the folded protein surface, but contribute to thermalinstability. Mutation of all three positions (e.g., Q40P, S47I, andH93G) results in a protein (FGF-1 3X) having enhanced thermostability.Mutation of all four positions (e.g., Q40P, S47I, H93G, and K112N)results in a protein (FGF-1 4X, FIG. 3B) that is at least as stable asFGF-1 bound with heparin (Zakrzewska, J. Biol. Chem. 284:25388-25403,2009).

Mutated FGF-1 induced ERK phosphorylation similar to the wild typeprotein (FIG. 4C). In contrast to wild type FGF-1, FGF-1 having theQ40P, S47I, H93G, and/or K112N mutation was able to maintain ERKphosphorylation after 24 hours (FIG. 3C). Heparin further increasedthermostability of FGF-1 3X (Q40P, S47I, and H93G) and FGF-2, while thestability of FGF-1 4X (Q40P, S47I, H93G, and K112N) was not furtherenhanced by heparin (FIG. 3D). The 3X and 4X mutated FGF-1 proteinsincreased ES cell growth and pluripotency (FIGS. 3E and 3F and 4D).Human ES cells maintained in media with FGF-1 3X and FGF-1 4X for 2months had normal karyotypes and gene expression characteristic of humanpluripotent cells (FIG. 4D). Truncation of FGF-1 does not impactthermostability of the protein (FIG. 4E).

Example 3 FGF Stability Affects Differentiation of Human PluripotentStem Cells

ES cells grown in culture in the absence of TGF-β express genesindicative of differentiation, such as GATA2 and HAND1. FGF-2 wassufficient to temporarily suppress the expression of specificdifferentiation marker genes, and heparin significantly enhanced thisfunction of FGF-1 3X and FGF-2 (FIGS. 5A and 5B) demonstrating thatthermostability influences the specific quality of FGF function.

To determine if FGF stability affects its ability to support mesodermaldifferentiation, FGF-1 K112N, 3X and 4X mutants were added to ES cellsand expression of the mesodermal marker Brachyury (T) and NANOG, whichis associated with Brachyury (T) expression, was analyzed. Human EScells differentiated into mesodermal lineage cells in the presence ofBMP4, as evidenced by their upregulated expression of Brachyury (T) andNANOG. Cells treated with thermostable FGF-1 also expressedsignificantly higher levels of NANOG (FIG. 5B) and Brachyury (T) (FIG.5C).

Example 4 FGF Stability Affects Reprogramming of Somatic Cells to HumanPluripotent Stem Cells

Methods

To determine if FGF thermostability affects reprogramming, foreskinfibroblasts were reprogrammed using a viral-free approach as previouslydescribed (Chen et al., 2011). Different FGFs (100 ng/ml) were used toreplace FGF-2 in culture media at each reprogramming stage. iPS colonieswere scored 30 days after the transfection of reprogramming factors(FIG. 6).

iPS Cell Derivation in Defined Conditions.

Briefly, plasmid combinations #19 (pEP4-E-O2S-E-T2K, pEP4-E-O2S-E-N2Kand pCEP4M2L) were used for reprogramming unless mentioned otherwise.Plasmids and EBNA mRNA were electroporated into fibroblast cells onAmaxa apparatus according to company instructions. One million cellswere used in each electroporation, which were then plated into two6-well plates. E8+ hydrocortisone media were used for the first 5-10days after electroporation. When confluency reached ˜20%, hydrocortisonewas removed. ES-like iPS cell colonies appeared after ˜25 days. Cellswere then picked into individual wells containing E8 medium withTGF-beta.

Results

Thermostable FGF-1 significantly improves cell growth of humanfibroblasts (FIG. 5E). Thermostable FGF-1 also significantly improvedreprogramming efficiency relative to truncated wild type FGF-1 (FIG. 5Fand FIG. 6).

SEQUENCES SEQ ID NO: 1: nucleic acid sequence oftruncated wild type human FGF-1ATGGCTAATTACAAGAAGCCCAAACTCCTCTACTGTAGCAACGGGGGCCACTTCCTGAGGATCCTTCCGGATGGCACAGTGGATGGGACAAGGGACAGGAGCGACCAGCACATTCAGCTGCAGCTCAGTGCGGAAAGCGTGGGGGAGGTGTATATAAAGAGTACCGAGACTGGCCAGTACTTGGCCATGGACACCGACGGGCTTTTATACGGCTCACAGACACCAAATGAGGAATGTTTGTTCCTGGAAAGGCTGGAGGAGAACCATTACAACACCTATATATCCAAGAAGCATGCAGAGAAGAATTGGTTTGTTGGCCTCAAGAAGAATGGGAGCTGCAAACGCGGTCCTCGGACTCACTATGGCCAGAAAGCAATCTTGTTTCTC CCCCTGCCAGTCTCTTCTGATTAASEQ ID NO: 2: amino acid sequence oftruncated wild type human FGF-1 (bold,underlined residues are Q40, S47, H93, and K112)MANYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSD Q HIQLQL SAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLE EN H YNTYISKKHAEKNWFVGL KKNGSCKRGPRTHYGQKAILFL PLPVSSD amino acid sequence of truncated wild typehuman FGF-1 (SEQ ID NO: 2) with Q40P, S47I, and H93G substitutionsSEQ ID NO: 3  MANYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSD P HIQLQL IAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLE EN GYNTYISKKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFL PLPVSSDamino acid sequence of truncated wild typehuman FGF-1 (SEQ ID NO: 2) with Q40P, S47I,H93G, and K112N substitutions SEQ ID NO: 4MANYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSD Q HIQLQL SAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLE EN H YNTYISKKHAEKNWFVGL NKNGSCKRGPRTHYGQKAILFL PLPVSSD

The invention has been described in connection with what are presentlyconsidered to be the most practical and preferred embodiments. However,the present invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,those skilled in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

The invention claimed is:
 1. A method for differentiating a humanpluripotent stem cell into a mesoderm lineage cell, the methodcomprising the step of: culturing the human pluripotent stem cell in afully defined culture medium comprising BMP4 and an FGF comprising theamino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 until the culturedcell expresses mesoderm lineage markers.
 2. The method of claim 1,wherein the FGF comprises the amino acid sequence of SEQ ID NO:3.
 3. Themethod of claim 2, wherein the amino acid sequence of the FGF consistsof the amino acid sequence of SEQ ID NO:3.
 4. The method of claim 1,wherein the amino acid sequence of the FGF comprises the amino acidsequence of SEQ ID NO:4.
 5. A method for reprogramming a human somaticcell into a human pluripotent stem cell, comprising: culturing a somaticcell expressing a plurality of reprogramming factors sufficient toinduce pluripotency in a fully defined culture medium comprising factorsthat support pluripotency of human pluripotent stem cells until the cellexpresses markers indicative of a human pluripotent stem cell, whereinthe medium comprises water, salts, amino acids, vitamins, glucose,insulin, selenium and an FGF comprising the amino acid sequence of SEQID NO:3 or SEQ ID NO:4.
 6. The method of claim 5, wherein the FGFcomprises the amino acid sequence of SEQ ID NO:3.
 7. The method of claim6, wherein the amino acid sequence of the FGF consists of the amino acidsequence of SEQ ID NO:3.
 8. The method of claim 5, wherein the FGFcomprises the amino acid sequence of SEQ ID NO:4.
 9. The method of claim8, wherein the amino acid sequence of the FGF consists of the amino acidsequence of SEQ ID NO:4.
 10. The method of claim 5, wherein the mediumfurther comprises heparin.